Antigen presentation begins with the association of small peptides and the major histocompatability complex (MHC) within antigen presenting cells (APCs). Recognition of specific non-self peptide-MHC complexes is central to one's ability to fight infection, while mistakenly recognizing self-peptide-MHC complexes as foreign is responsible for autoimmune pathology such as is found in type-1 diabetes. Current autoimmune therapies are not specific, and thus complicated by the side-effects of general immune suppression. There is little doubt that a better understanding of the differences in these two processes will lead to more specific therapies. The cellular organelle in which peptide-MHC complexes form, the processes guiding their transport to the cell surface, and the mechanisms controlling their longevity are all unknown because it has heretofore been impossible to visualize the formation of peptide-MHC complexes in live cells with high resolution. This proposal applies a newly developed technique - bipartite tetracysteine display - to image and track the formation of peptide-MHC complexes in live cells. In bipartite tetracysteine display, pairs of cysteines on each of two binding partners become closely juxtaposed upon complex formation. This newly formed tetracysteine motif is rapidly and specifically labeled by FIAsH and ReAsH. For this proposal, we will first (AIM 1), engineer peptide-MHC complexes for split tetracysteine display and evaluate their ability to bind FIAsH and ReAsH in complex but not separately. We will then (AIM 2), analyze expression of these proteins in live cells and track the spatial and temporal regulation of peptide-MHC complex formation. Finally (AIM 3), we will exploit the ability to track antigen presentation in live cells to screen a library of small molecule and peptide ligands for the ability to modulate antigen presentation. The ability to visualize and track peptide-MHC complexes has the potential to address questions concerning the biological mechanism of antigen presentation. Eventually, the ability to block the formation of complexes responsible for autoimmunity could be a more specific approach to the treatment of autoimmunity. PUBLIC HEALTH RELEVANCE Split tetracysteine display achieves spatial and temporal resolution over what can be achieved using fluorescent proteins or antibodies. This proposal focuses on the development of imaging tools capable of specifically detecting proteins and protein complexes without the use fluorescent proteins or antibodies through the use of 'pro-fluorescent'small molecules that act as 'turn-on'fluorescent sensors for particular chemical structures. These studies will improve our understanding of normal and disease physiology and guide the development of new therapies.